We have data for 2 FL samples: FL1018T1 and FL1018T2. This patient has transformed FL.
We filter on a mitochondrial threshold of 10% and a ribosomal threshold of 60%. These thresholds were indicated in the previous plots.
We normalize by:
scran (after quickCluster)scater::normalizeWe use cyclone to assign cells to cell cycle states.
| B cells | T cells | |
|---|---|---|
| CD19 | 1 | 0 |
| CD28 | 0 | 1 |
| CD3D | 0 | 1 |
| CD3E | 0 | 1 |
| CD3G | 0 | 1 |
| CD74 | 1 | 0 |
| CD79A | 1 | 0 |
| CD79B | 1 | 0 |
| IGKC | 1 | 0 |
| IGLC2 | 1 | 0 |
| IGLC3 | 1 | 0 |
| MS4A1 | 1 | 0 |
| TRAC | 0 | 1 |
| B cells | T cells |
|---|---|
| 3452 | 688 |
| B cells | Cytotoxic T cells (activated) | Cytotoxic T cells | Naive/resting CD4 | CD4 (activated) | Tfh | other | |
|---|---|---|---|---|---|---|---|
| CCL5 | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| CCR7 | 1 | 0 | 0 | 1 | 0 | 0 | 0 |
| CD19 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| CD2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
| CD3D | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
| CD3E | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
| CD3G | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
| CD4 | 0 | 0 | 0 | 1 | 1 | 1 | 0 |
| CD69 | 0 | 1 | 0 | 0 | 1 | 1 | 0 |
| CD74 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| CD79A | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| CD79B | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| CD8A | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| CD8B | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| CXCR5 | 1 | 0 | 0 | 0 | 0 | 1 | 0 |
| EOMES | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| GZMA | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| ICA1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| ICOS | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| IFNG | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| IGKC | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| IGLC2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| IGLC3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| IL7R | 0 | 0 | 0 | 1 | 1 | 0 | 0 |
| MS4A1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| NKG7 | 0 | 1 | 1 | 0 | 0 | 0 | 0 |
| PDCD1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| SELL | 1 | 0 | 0 | 1 | 0 | 0 | 0 |
| ST8SIA1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| TNFRSF4 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| TRAC | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
We can look for subclusters of B cells, e.g. memory vs. naive, within the malignant population.
Similar results for other IgG chains. No IgM, IgD, or IgA expression.
For whatever reason, T1 cells express IgE.
NOTE: This is predominantly present in T2 as opposed to T1. These cells correspond to the S phase cells observed in T2.
What’s going on in this cluster?
We can look for subclusters of B cells, e.g. memory vs. naive, within the nonmalignant population.
We don’t really have enough cells here to do anything meaningful.
We’ll look to see how celltype proportions change between timepoints.
| FL1018T1 | FL1018T2 | |
|---|---|---|
| B cells | 7.65 | 0.44 |
| B cells (malignant) | 56.98 | 92.34 |
| CD4 (activated) | 4.91 | 1.80 |
| Cytotoxic T cells | 4.21 | 0.70 |
| Cytotoxic T cells (activated) | 2.32 | 2.95 |
| Naive/resting CD4 | 8.77 | 0.33 |
| other | 0.35 | 0.07 |
| Tfh | 14.81 | 1.36 |
| FL1018T1 | FL1018T2 | |
|---|---|---|
| Cytotoxic T cells (activated) | 6.61 | 41.24 |
| Cytotoxic T cells | 12.02 | 9.79 |
| Naive/resting CD4 | 25.05 | 4.64 |
| CD4 (activated) | 14.03 | 25.26 |
| Tfh | 42.28 | 19.07 |
| FL1018T1 | FL1018T2 | |
|---|---|---|
| B cells | 11.83 | 0.48 |
| B cells (malignant) | 88.17 | 99.52 |
The nonmalignant fraction of B cells becomes virtually nonexistant in T2.
We’ll look at genes that are differentially expressed between malignant and nonmalignant B cells. We treat timepoint as a blocking variable.
In the plots that follow, genes that are upregulated in malignant cells relative to nonmalignant cells have positive logFC values.
Some key genes for which we see consistent observations are BCL2, BCL6, and ID-2.
Refer to http://www.bloodjournal.org/content/99/1/282 for a more comprehensive list of markers to check.
For each celltype, we ask what genes and pathways are differentially expressed between timepoints. In the plots that follow, genes that are upregulated in T2 relative to T1 will have positive logFC values.
Upregulation of CD8 T effector activity in T2?
Downregulation in antigen presentation – perhaps due to corresponding upregulation of T effector response in T2?
Nothing significant.
Looks like heat shock proteins HSPA1A, HSPA1B, and HSPH1 are downregulated in T2. May be an artifact of processing. We can see if this is consistent with other celltypes. Note that CD69 is also upregulated in T2 relative to T1 – activation of CD4 T cells in T2 as well. We need to give a thought to the CXCR4 population though. Are those Tfh’s or just T mem’s? Not well described in the literature as far as I’m aware.
Nothing significant.
Again, we see the heat shock response downregulated in T2 relative to T1. However, JUN is also upregulated in T2 – could be biological or batch-associated. Once again – CD69 is upregulated in T2.
Nothing significant. However, do notice that the number of nonmalignant B cells has dropped quite precipitously, especially in relation to the discordant rise in malignant B cell prevalence.
No HLA loss or anything of much interest here, I think. TCL1A and FCER2 might be interesting though, with more FL knowledge.
| G1 | G2M | S | |
|---|---|---|---|
| B cells | 77 | 6 | 38 |
| B cells (malignant) | 1844 | 148 | 1327 |
| CD4 (activated) | 90 | 10 | 19 |
| Cytotoxic T cells | 56 | 4 | 19 |
| Cytotoxic T cells (activated) | 77 | 9 | 27 |
| Naive/resting CD4 | 87 | 9 | 38 |
| other | 6 | 0 | 1 |
| Tfh | 155 | 23 | 70 |
| G1 | G2M | S | |
|---|---|---|---|
| B cells | 63.64 | 4.96 | 31.40 |
| B cells (malignant) | 55.56 | 4.46 | 39.98 |
| CD4 (activated) | 75.63 | 8.40 | 15.97 |
| Cytotoxic T cells | 70.89 | 5.06 | 24.05 |
| Cytotoxic T cells (activated) | 68.14 | 7.96 | 23.89 |
| Naive/resting CD4 | 64.93 | 6.72 | 28.36 |
| other | 85.71 | 0.00 | 14.29 |
| Tfh | 62.50 | 9.27 | 28.23 |
As expected (perhaps), cancer cells have the highest percentage of cycling cells. Overall though, we may be overestimating the percentage of cycling cells – check and see what is going on with cyclone here.
| G1 | G2M | S | |
|---|---|---|---|
| FL1018T1 | 928 | 80 | 417 |
| FL1018T2 | 1464 | 129 | 1122 |
| G1 | G2M | S | |
|---|---|---|---|
| FL1018T1 | 65.12 | 5.61 | 29.26 |
| FL1018T2 | 53.92 | 4.75 | 41.33 |
There’s a higher percentage of cycling cells in T2 – consistent with disease transformation and worsening patient prognosis.
We can go deeper into investigating this and see if ALL cells in T2 have higher cycling percentages, or only cancer cells.
Fixed effects: timepoint, celltype Interaction terms: none (a timepoint*celltype interaction term would overparametrize)
Response family: binomial
Normality testing is unnecessary as we do not assume it (and it is generally useless to begin with).
##
## Call:
## glm(formula = cbind(cycling_count, total_count - cycling_count) ~
## dataset + celltype_full, family = "binomial", data = cycling_summary)
##
## Deviance Residuals:
## Min 1Q Median 3Q Max
## -1.67774 -1.02843 0.00414 0.60776 2.44142
##
## Coefficients:
## Estimate Std. Error z value
## (Intercept) -0.60241 0.18952 -3.179
## datasetFL1018T2 0.41404 0.07648 5.414
## celltype_fullB cells (malignant) 0.06410 0.19909 0.322
## celltype_fullCD4 (activated) -0.71119 0.28731 -2.475
## celltype_fullCytotoxic T cells -0.39328 0.31269 -1.258
## celltype_fullCytotoxic T cells (activated) -0.45773 0.28154 -1.626
## celltype_fullNaive/resting CD4 -0.04250 0.26214 -0.162
## celltype_fullother -1.32038 1.09929 -1.201
## celltype_fullTfh 0.02755 0.23057 0.119
## Pr(>|z|)
## (Intercept) 0.00148 **
## datasetFL1018T2 6.17e-08 ***
## celltype_fullB cells (malignant) 0.74749
## celltype_fullCD4 (activated) 0.01331 *
## celltype_fullCytotoxic T cells 0.20849
## celltype_fullCytotoxic T cells (activated) 0.10399
## celltype_fullNaive/resting CD4 0.87120
## celltype_fullother 0.22970
## celltype_fullTfh 0.90491
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## (Dispersion parameter for binomial family taken to be 1)
##
## Null deviance: 91.133 on 15 degrees of freedom
## Residual deviance: 17.761 on 7 degrees of freedom
## AIC: 102.7
##
## Number of Fisher Scoring iterations: 4
Coefficients are going the right way. Not exactly surprising that most of the coefficients are nonsignificant – after all, we don’t have many data points at all.
An observation we may want to note is how the percentage of cycling malignant B cells vs. other celltypes changes from T1 to T2.
| G1 | G2M | S | |
|---|---|---|---|
| B cells | 65.14 | 2.75 | 32.11 |
| B cells (malignant) | 64.90 | 4.93 | 30.17 |
| CD4 (activated) | 74.29 | 7.14 | 18.57 |
| Cytotoxic T cells | 71.67 | 3.33 | 25.00 |
| Cytotoxic T cells (activated) | 54.55 | 9.09 | 36.36 |
| Naive/resting CD4 | 65.60 | 6.40 | 28.00 |
| other | 100.00 | 0.00 | 0.00 |
| Tfh | 61.61 | 9.00 | 29.38 |
| G1 | G2M | S | |
|---|---|---|---|
| B cells | 50.00 | 25.00 | 25.00 |
| B cells (malignant) | 52.53 | 4.31 | 43.16 |
| CD4 (activated) | 77.55 | 10.20 | 12.24 |
| Cytotoxic T cells | 68.42 | 10.53 | 21.05 |
| Cytotoxic T cells (activated) | 73.75 | 7.50 | 18.75 |
| Naive/resting CD4 | 55.56 | 11.11 | 33.33 |
| other | 50.00 | 0.00 | 50.00 |
| Tfh | 67.57 | 10.81 | 21.62 |
So, the only celltype for which we have a decent number of cells (see next slide) and have a substantial increase in cycling cells is in the B cell population. So indeed, the B cell population is becoming more actively cycling in T2 while the other populations aren’t.
| celltype_full | count |
|---|---|
| B cells | 12 |
| B cells (malignant) | 2507 |
| CD4 (activated) | 49 |
| Cytotoxic T cells | 19 |
| Cytotoxic T cells (activated) | 80 |
| Naive/resting CD4 | 9 |
| other | 2 |
| Tfh | 37 |